Angiogenesis initiation and growth

ABSTRACT

Methods for promoting angiogenesis comprising administering platelet-rich plasma to a site and stimulating the site with an electromagnetic field. Platelets include platelet-rich plasma and compositions can further include stem cells such as adipose stromal cells and cells derived from bone marrow aspirate. Methods also comprise isolating platelets from a patient&#39;s blood, forming a composition including the platelets, delivering the composition to a site in need of treatment, and electrically stimulating the site.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/714,520 filed Mar. 6, 2007, the disclosure of which is incorporatedherein by reference.

INTRODUCTION

The present technology relates to methods for promoting angiogenesis,including vascularizing an avascular site, or extending or enhancingexisting vasculature.

Angiogenesis is generally referred to as the growth of new and/orextension of preexisting blood vessels and is an important naturalprocess in the body. Angiogenesis occurs following an injury as part ofnormal wound healing to restore blood flow to damaged tissues. Methodsand compositions that promote and enhance angiogenesis are desirable andwould prove beneficial in treating damaged or diseased tissue.

SUMMARY

The present technology provides a method for promoting angiogenesis at asite on or within a patient. In some embodiments, the method comprisesobtaining blood compatible with the patient and isolating platelet-richplasma by centrifugation of the blood. The platelet-rich plasma isadministered to the site, and then the site is stimulated with anelectromagnetic field. Angiogenesis is thereby promoted at the site dueto application of the platelet-rich plasma and the electromagneticfield.

Further areas of applicability of the present teachings will becomeapparent from the detailed description provided herein. It should beunderstood that the detailed description and specific examples, whileindicating various embodiments of the teachings, are intended forpurposes of illustration only and are not intended to limit the scope ofthe teachings.

BRIEF DESCRIPTION OF THE DRAWINGS

The present teachings will become more fully understood from thedetailed description and the accompanying drawings, wherein:

FIG. 1 illustrates a representative site on a patient in which thepromotion of angiogenesis is desirable according to one embodiment ofthe present teachings;

FIG. 2 is a schematic illustration of a representative method forpromoting angiogenesis according to one embodiment of the presentteachings;

FIG. 3 is a cross-sectional view of a representative device used forisolating platelet-rich plasma according to one embodiment of thepresent teachings;

FIG. 4 illustrates a representative manner of administratingplatelet-rich plasma to the patient according to one embodiment of thepresent teachings; and

FIG. 5 illustrates a representative manner for stimulating the site onthe patient with an electromagnetic field according to one embodiment ofthe present teachings.

DESCRIPTION

The description of the following technology is merely exemplary innature of the subject matter, manufacture, and use of the teachingdisclosed herein, and is not intended to limit the scope, application,or uses of any specific invention claimed in this application, or insuch other applications as may be filed claiming priority to thisapplication, or patents issuing therefrom.

Referring to FIG. 1, a soft tissue wound 10 on a patient's foot 12 isshown. The soft tissue wound may be an ulcer (e.g., a venous ulcer,pressure ulcer, or diabetic ulcer), and it may be located on a bodyextremity or elsewhere on a patient's body, such as on the torso orhead. While the following discussion uses a soft tissue wound on a footas a representative example of a site on a patient where angiogenesis isdesired, other suitable locations and conditions exist. Some examples ofsites where promoting angiogenesis is beneficial include locationsexhibiting the following conditions: coronary artery disease, stroke,delayed wound healing, chronic wounds, peripheral vascular disease,ischemia, chronic tendonosis, wounds or tissue breakdown resulting fromradiation treatment, and myocardial infarction.

One embodiment of the method for promoting angiogenesis is showndiagrammatically in FIG. 2. In summary, blood may be initially drawnfrom the patient at step 14. Alternatively, donor blood identified ascompatible with the patient may be obtained as shown by step 16. At step18, platelet-rich plasma is isolated from the blood of the patientand/or the blood donor. After the platelet-rich plasma has been isolatedat step 18, optional materials, such as stem cells, angiogenic factors,platelet activators, and scaffold materials, may be added at step 20.Platelet-rich plasma, with one or more optional materials if desired, isthen administered to the site at step 22, and then the site isstimulated with an electromagnetic field at step 24. The platelet-richplasma and the electromagnetic stimulation act in concert to moreeffectively promote angiogenesis at the site than when the componentsare used individually. In this regard, administration of platelet-richplasma, together with stimulating the site with an electromagneticfield, results in more complete vascularization and/orre-vascularization of the site compared to vascularization achievedusing either step alone. Each of the steps identified above will be morefully discussed below.

As discussed above, blood compatible with the patient is initiallyobtained so that platelet-rich plasma may be isolated. This can includedrawing the patient's own blood as indicated by step 14 and/or obtainingdonor blood compatible with the patient as indicated by step 16. Bloodfrom the patient can be obtained as needed or can be obtained hours,days, or several weeks in advance of the treatment, based on bloodstorage and preservation methods as generally practiced in the art. Ifdonor blood is to be used, compatible donor blood can be identifiedusing standard blood tests, including, for example, matching blood cellsurface antigens.

Once blood is obtained from the patient at step 14 and/or from a donorat step 16, platelet-rich plasma is isolated at step 18. Platelet-richplasma can be isolated from the blood obtained in steps 14 or 16 by oneor more techniques including filtration, and density fractionationmethods such as centrifugation of whole blood, centrifugation of bloodin multiple stages, and continuous-flow centrifugation. One example of adevice that may be used in isolating platelet-rich plasma is shown inFIG. 3. In this regard, blood obtained in either steps 14 or 16 isinitially loaded into the device 26 having a tube 28 with a buoy 30located within the tube 28. The device 26 is then centrifuged so thatthe buoy 30 defines an interface 32 between platelet-rich plasma 34 andplatelet poor plasma 36. The remaining red blood cells 38 sediment belowthe buoy 30. Once the platelet-rich plasma 34 has been isolated by thebuoy 30 during centrifugation, the platelet-rich plasma 34 can becollected and applied to the patient as discussed below.

Embodiments of such devices used for isolating platelet-rich plasmainclude the GPS® II Platelet Concentrate Separation Kit and the Plasmax™Plus Plasma Concentrator accessory (Biomet Biologics, Inc., Warsaw,Ind.). Such devices and methods are described in U.S. Patent ApplicationPublication 2004/0251217 (Leach et al.), and U.S. Patent ApplicationPublication 2005/0109716 (Leach et al.), which are hereby incorporatedby reference.

Another example of a device that may be used in isolating platelet-richplasma by density frationation includes a centrifugal drum separator andan erythorocyte capture trap. In one embodiment, the walls of thecentrifugal drum separator are coated with a depth filter having poresand passageways that are sized to receive and entrap erythrocytes. Bloodis placed in the centrifugal drum, and the drum is spun along its axisat sufficient speed so as to force erythrocytes from the blood into thedepth filter. After spinning, the erythrocytes remain in the filter andthe remaining platelet-rich plasma is extracted. The platelet-richplasma may be concentrated by desiccation. Embodiments of such devicesinclude the Vortech™ Concentration System (Biomet Biologics, Inc.,Warsaw, Ind.), and are disclosed in U.S. Patent Application Publication2006/0175244 (Dorian et al.) and U.S. Patent Application Publication2006/0175242 (Dorian et al.), which are hereby incorporated byreference. Such devices may be used to prepare platelet-rich plasma inlieu of or in addition to using the tube having a buoy that is describedabove and shown in FIG. 2.

Other devices for isolating platelet-rich plasma use high speedcentrifugation to pellet the platelets and red blood cells. The pelletedplatelets are then resuspended using some of the plasma supernatant oranother suitable solution. It will be understood, however, that othersuitable methods for forming the platelet-rich plasma 34 may also beused.

The concentration of platelets within the platelet-rich plasma 34 mayvary. For example, in some embodiments, the platelet concentration inthe platelet-rich plasma 34 can be from about 3-fold to about 10-foldgreater than the platelet concentration in whole blood. Furthermore, theplatelet-rich plasma 34 can contain cytokines, growth factors, and otherproteins and molecules in addition to those contained within orfractionating with the platelets. For example, pelleted platelets thatare resuspended in whole or in part with a plasma supernatant cancontain cytokines and growth factors from the plasma supernatant.

Referring again to FIG. 2, optional materials, such as stem cells,angiogenic factors, platelet activators, and scaffold materials, may beused with the platelet-rich plasma 34 as indicated by step 20. Withrespect to the use of stem cells, the source of the stem cells may beadipose stromal cells and/or stem cells derived from bone marrowaspirate. The stem cells can be mammalian stem cells, and in variousembodiments, are human stem cells. Adipose stromal cells may be derivedfrom adult adipose tissue harvested by lipoaspiration or liposuction.Such methods include those disclosed in U.S. Pat. No. 6,355,239 and U.S.Pat. No. 6,541,024. Stem cells derived from bone marrow aspirate can beharvested by needle aspiration of bone marrow and may be harvested fromthe posterior illiac crest, anterior illiac crest, sternum or tibia. Thestem cells may be autologous cells, allogenic cells, or xenogeneiccells. Preferably, the stem cells are autologous or allogenic. Stemcells can be applied to the site just prior to the administration of theplatelet-rich plasma 34, concomitant with administration theplatelet-rich plasma 34, or following administration of theplatelet-rich plasma 34 to the patient.

In some embodiments, isolation of stem cells can be performed byextraction of tissue by standard lipoaspiration, isolation from excisedadipose tissue, or by using the VASER® ultrasound disruptor incombination with the VENTX™ cannula, available from Sound SurgicalTechnologies, LLC, Louisville, Colo. The GPS™ biomaterial separationunit (Biomet Biologics, Inc., Warsaw, Ind.) can also be used to isolateadipose stem cells from aspirated adipose tissue. Such devices andmethods are described in U.S. Patent Application Publication2004/0251217 and U.S. Patent Application Publication 2005/0109716 whichare hereby incorporated by reference.

Step 20 may also involve the addition or use of platelet activators. Theplatelet activators serve to release the growth factors within theplatelets forming the platelet-rich plasma 34. Platelet activators mayinclude thrombin, calcium chloride, collagen, and mixtures thereof.Activation of the platelets in platelet-rich plasma 34 by the plateletactivators can occur just prior to administration of the platelet-richplasma 34 to the patient, concomitant with administration theplatelet-rich plasma 34 to the patient, or following administration ofthe platelet-rich plasma 34 to the patient.

Step 20 can also involve the use of various angiogenic factors.Angiogenic factors can include, but are not limited to, the followingmolecules: angiogenin, angiopoietin-1, del-1 protein, fibroblast growthfactors such as acidic FGF (also known as aFGF or FGF-1) and basic FGF(also known as bFGF or FGF-2), follistatin, granulocytecolony-stimulating factor (G-CSF), hepatocyte growth factor (HGF),interleukin-8 (IL-8), leptin, midkine, placental growth factor,platelet-derived endothelial growth factor (PD-ECGF), platelet-derivedgrowth factor (PDGF), pleiotrophin (PTN), progranulin, proliferin,transforming growth factor alpha (TGF-α), transforming growth factorbeta (TGF-β), tumor necrosis factor alpha (TNF-α), vascular endothelialgrowth factor (VEGF), and vascular permeability factor (VPF). In variousembodiments, isolated, recombinant, and/or synthetic angiogenic factorsmay be used. Angiogenic factors can be applied to the site just prior tothe administration of the platelet-rich plasma 34, concomitant withadministration the platelet-rich plasma 34, or following administrationof the platelet-rich plasma 34 to the patient.

A scaffold may be included as indicated at step 20 to contain, support,or retain the platelet-rich plasma or other materials used in thepresent methods at the tissue defect site. For example, a scaffold maycontain or support stem cells that may be included in step 20,preferably enabling growth and/or retention of the stem cells at thesite of implantation. The scaffold may also provide a support forretention of the platelet-rich plasma at the wound site. In addition,the scaffold may facilitate migration of endogenous cells into theadministration site. The scaffold may be implanted or applied at thetissue defect site, followed by the administration of platelet-richplasma and optional materials in step 22. Alternatively, theplatelet-rich plasma or optional material may be combined with thescaffold prior to administration in step 22.

Scaffolds may be formed from porous or semi-porous, natural, syntheticor semisynthetic materials. In some wound treatment applications, ascaffold material can be an osteoconductive material. Scaffold materialsinclude those selected from the group consisting of bone (includingcortical and cancellous bone), demineralized bone, ceramics, polymers,and combinations thereof. Suitable polymers may include collagen,including lyophilized or skin-derived collagen as disclosed in U.S.patent application Ser. No. 11/259,216 which is hereby incorporated byreference. Polymers may also include, gelatin, hyaluronic acid,chitosan, polyglycolic acid, polylactic acid, polypropylenefumarate,polyethylene glycol, and copolymers or combinations thereof. Ceramicsinclude any of a variety of ceramic materials known in the art for usefor implanting in bone, such as calcium phosphate (including tricalciumphosphate, tetracalcium phosphate, hydroxyapatite, and mixturesthereof). Ceramics useful herein include those described in U.S. Pat.No. 6,323,146 and U.S. Pat. No. 6,585,992. A commercially availableceramic is ProOsteon™ from Interpore Cross International, Inc. (Irvine,Calif.).

After the platelet-rich plasma 34 has been isolated at step 18, it isadministered to the site of a tissue defect in step 22, optionally withone or more optional materials such as platelet activators, stem cells,angiogenic factors, buffering agents and scaffold materials as may beadded in step 20. When such optional materials are used, theplatelet-rich plasma 34 and one or more optional materials can beadministered either together as a single composition, or theplatelet-rich plasma and each material can be administered separately orin various sub-combinations.

In certain forms of treatment, the platelet-rich plasma 34 may beapplied to an external surface of the patient. For example, as shown inFIG. 4, the platelet-rich plasma 34 can be sprayed onto the site of askin wound 10 using a spray applicator 40 containing the platelet-richplasma 34.

In other forms of treatment, the platelet-rich plasma 34 may be appliedat a site internal to the patient. In this regard, platelet-rich plasma34 can be injected into internal tissue or organs by means of a syringe.In addition, platelet-rich plasma 34 can be administered in conjunctionwith a surgical procedure. The administration of the platelet-richplasma 34 to an site internal to the patient can be used to treat, forexample, the effects of coronary artery disease, stroke, delayed woundhealing, chronic wounds, peripheral vascular disease, ischemia, chronictendonosis, wounds or tissue breakdown resulting from radiationtreatment, and myocardial infarction. It should be understood, however,that the step of administering platelet-rich plasma 34 may also compriseany biomedically acceptable process or procedure by which theplatelet-rich plasma 34 is implanted, injected, or otherwiseadministered in, on, or in proximity to the site on a patient so as tohave a beneficial effect by promoting angiogenesis.

Following the application of platelet-rich plasma 34 to the site at step22, the site is stimulated with an electromagnetic field as indicated bystep 24. Stimulating the site with an electromagnetic field may involvevarious forms of electromagnetic stimulation. In one embodimentillustrated in FIG. 5, the site is stimulated using a power source 42coupled to a stimulation coil 44. The current passing through the coilproduces a pulsing magnetic field which induces in the site to betreated a pulsing electric field. The coil 44 may be a saddle-shapedcoil that at least partially surrounds a wound site on the patient'sfoot 12 and the adjacent tissue, and it may be held in place with abandage, tape, Velcro® or other suitable fasteners, or as part of apadded boot 46. The coil 44 is connected via a cable 48 to the powersource 42 which may be held against the patient's body (e.g., on thethigh or on the waist) in any suitable manner.

Step 24 involving the stimulation of a site with an electromagneticfield may also include placing at least two electrodes across the siteto be treated on the patient. Electrical energy may then be applied tothe electrodes so as to capacitively couple the electrodes and generatethe electromagnetic field therebetween. The electromagnetic field istherefore able to pass through the platelet-rich plasma 34 so as topromote angiogenesis at the site by upregulation of angiogenic factors,including FGF-2, and extension of preexisting and/or growth of new bloodvessels. In other embodiments, step 24 may also involve implantingelectrodes to produce a direct current, or one or more coils, into thepatient and administering an electric stimulation to the site via theimplanted electrodes or coil(s).

The strength of the electromagnetic field produced in the tissue duringelectrical stimulation is preferably at least about 0.5 microvolts percentimeter whether produced by direct current, capacitive coupledcurrent or pulsed electromagnetic fields. In the case of an implanteddirect current electrode, the amplitude of the current should be fromabout 1 to about 200 microamperes in some embodiments, and in otherembodiments, the amplitude may be from about 20 to about 100microamperes. In still further embodiments, the current may be about 20,about 60, or about 100 microamperes. It should be understood, however,that the amplitude of the current may be of other suitable magnitudes.

The electromagnetic field applied at step 24 may be constant or varyover time as discussed above. For example, a sinusoidal time varyingelectromagnetic field can be applied by the electrodes placed across thesite. Such a sinusoidal time varying electromagnetic field can have apeak voltage across the electrodes from about 1 volt to about 10 volts,and in some embodiments, the peak voltage can be about 5 volts. Thecorresponding electric field produced in the tissue can have anamplitude of from about 0.1 millivolt per centimeter (mV/cm) to about100 mV/cm, and in some embodiments can be about 20 mV/cm. The sinusoidaltime varying electric field may have a frequency of from about 1,000 Hzto about 200,000 Hz, and in some embodiments the frequency may be about60,000 Hz.

The electromagnetic field applied to the site at step 24 may also be apulsed electromagnetic field. In this regard, a pulsed electromagneticfield may have a pulse duration of from about 10 microseconds per pulseto about 2000 microseconds per pulse. In addition, the pulse duration ofthe pulsed electromagnetic field in one embodiment can be about 225microseconds. The pulses may include in electromagnetic “bursts”, inwhich a burst can comprise from 1 pulse to about 200 pulses.Alternatively, the electromagnetic field may have bursts that comprisefrom about 10 pulses to about 30 pulses. In this regard, in oneembodiment each burst may comprise about 20 pulses.

The frequency at which bursts in the pulsed electromagnetic are appliedmay vary. In this regard, bursts can be repeated at a frequency of fromabout 1 Hz to about 100 Hz in some embodiments, and can be repeated at afrequency of about 10 Hz to about 20 Hz in other embodiments. Further,bursts can repeat at a frequency of about 1.5 Hz, about 15 Hz or about76 Hz. A burst can have a duration from about 10 microseconds up toabout 40,000 microseconds. In this regard, a burst can have a durationof about 4.5 milliseconds.

Suitable devices for generating a capacitively coupled electromagneticfield include SpinalPak® spinal stimulator (EBI, L.P., Parsippany, N.J.)or a DC stimulation device such as an SpF® XL IIb spinal fusionstimulator (EBI, L.P., Parsippany, N.J.). Pulsed electromagnetic fieldscan be produced using various known methods and apparatuses, such asusing a single coil or a pair of Helmholtz coils. For example, asuitable apparatus includes the EBI Bone Healing System® Model 2001(EBI, L.P., Parsippany, New Jersey). With respect to electricalstimulation of internal sites in a patient, a direct current electricfield may be generated using any known device for generating a directcurrent electric field, such as for example, the Osteogen™ implantablebone growth stimulator (EBI, L.P., Parsippany, N.J.). Other suitabledevices for generating electromagnetic fields may be used.

Electromagnetic stimulation of the site at step 24 can be continuedand/or repeated as necessary after the platelet-rich plasma 34 (and/orthe various compositions described herein) has been applied to thepatient. It should be understood, however, that the step of stimulatingthe site with an electromagnetic field includes fields other than, or inaddition to, electric or electromagnetic fields associated with ambientconditions (such the electromagnetic fields generated by casual exposureto radios, telephones, desktop computers or similar devices).

Specific examples are provided for illustrative purposes of how to makeand use the compositions and methods of this technology and, unlessexplicitly stated otherwise, are not intended to be a representationthat given embodiments of this technology have, or have not, been madeor tested.

Example 1

A patient presenting a chronic wound is treated using a method of thepresent technology. Platelet-rich plasma is prepared from a patient'sblood using the GPS® II Platelet Concentrate Separation Kit and thePlasmax™ Plus Plasma Concentrator accessory (Biomet Biologics, Inc.,Warsaw, Ind.). Adipose stromal cells are harvested from the patient byperforming lipoaspiration to obtain adipose tissue. The adipose tissueis enzymatically digested in collagenase type I solution (WorthingtonBiochemical) under gentle agitation for 1 hour at 37° C., thedissociated cells are filtered with 500-μm and 250-μm Nitex filters, andcentrifuged at 200 g for 5 minutes to separate the stromal cell fraction(pellet) from the adipocytes. The platelet-rich plasma is combined withthe isolated adipose stromal cells.

The platelet-rich plasma and adipose stromal cells are administered tothe patient's chronic wound site. Electric stimulation in the form of apulsed electromagnetic field is applied across the site using astimulation coil. The pulse duration of the pulsed electromagnetic fieldis about 225 microseconds per pulse. The pulses are comprised ofelectromagnetic bursts in which each burst contains about 20 pulses.Each burst is repeated at a frequency of about 15 Hertz and has aduration of about 4.5 milliseconds. The platelet-rich plasma, stemcells, and electric stimulation promote angiogenesis and achieve morecomplete healing of the patient's chronic wound by enhancingvascularization of the site in comparison with a similar wound leftuntreated.

Example 2

Tissue damaged by chronic ischemia due to peripheral artery occlusivedisease is treated by a method of the present disclosure. A compositionof platelet-rich plasma is injected to the ischemic site followed byelectromagnetic stimulation using direct current.

Allogenic platelets are purified from donated whole blood by high speedcentrifugation to sediment the platelets from the plasma. Pelletedplatelets are resuspended using a fraction of the plasma supernatant toform a viscous composition of platelet-rich plasma containing abouteight times greater concentration of platelets compared to whole blood.The platelet composition is administered to the ischemic site andelectromagnetic stimulation is provided across the site usingtemporarily implanted electrodes. The method promotes angiogenesis andresults in revascularization of the ischemic site.

Example 3

A process for promoting angiogenesis is used to promote angiogenesis ina patient's hand exhibiting chronic tendonosis. Blood is drawn from thepatient and platelet-rich plasma is isolated from the blood bycentrifugation and collection of the buffy coat fraction.

The platelet-rich plasma is administered to the site of the tendonosisby injection and the site is subjected to electromagnetic stimulationcomprising a pulsed electromagnetic field by using a pair of Helmholtzcoils. The composition promotes angiogenesis where new blood vesselsform to supply oxygenated blood to the site.

Example 4

A method for treating a patient with unilateral ischaemia of the legincludes injection of platelet-rich plasma and stem cells derived frombone marrow aspirate (BMA). Blood is drawn from the patient andplatelet-rich plasma is isolated using the GPS® II Platelet ConcentrateSeparation Kit (Biomet Biologics, Inc., Warsaw, Ind.). BMA is harvestedfrom the patient using standard techniques. Stem cells derived from theBMA are concentrated and isolated by centrifugation using the GPS® IIdevice. Both the platelet-rich plasma and the stem cells are isolatedand combined in the presence of a citrate-based anticoagulant.

The platelet-rich plasma and the stem cells derived from BMA areadministered to the patient around the site of vascular occlusion usingintramuscular injection. Between about 1×10⁹ and about 3×10⁹ stem cellsderived from BMA are combined with platelet-rich plasma in a totalvolume of about 30 mL. About 0.75 mL of is administered to 40 injectionsites in a 3×3 cm grid using a 26-gauge needle at a depth of about 1.5cm.

Electric stimulation in the form of a pulsed electromagnetic field isapplied to the site using a stimulation coil. The pulse duration of thepulsed electromagnetic field is about 225 microseconds per pulse. Thepulses are comprised of electromagnetic bursts in which each burstcontains about 20 pulses. Each burst is repeated at a frequency of about15 Hertz and has a duration of about 4.5 milliseconds. The platelet-richplasma, stem cells derived from BMA, and electric stimulation promoteangiogenesis at the site and increase the pain-free walking time of thepatient.

The examples and other embodiments described herein are exemplary andnot intended to be limiting in describing the full scope of compositionsand methods of this technology. Equivalent changes, modifications andvariations of specific embodiments, materials, compositions and methodsmay be made within the scope of the present technology, withsubstantially similar results.

What is claimed is:
 1. A method for promoting angiogenesis at a site ona patient, the method comprising: obtaining blood compatible with thepatient; isolating platelet-rich plasma by density fractionation of saidblood; administering said platelet-rich plasma to the site; andstimulating the site with an electromagnetic field followingadministration of said platelet-rich plasma to the site, therebypromoting angiogenesis at the site upon application of saidelectromagnetic field and said platelet-rich plasma to the site.
 2. Themethod for promoting angiogenesis at a site on a patient as set forth inclaim 1, wherein isolating platelet-rich plasma by density fractionationcomprises centrifuging of said blood by: loading said blood into a tubehaving a buoy; spinning said tube so that said buoy defines an interfacebetween platelet-rich plasma and platelet poor plasma contained withinsaid blood; and collecting said platelet-rich plasma.
 3. The method forpromoting angiogenesis at a site on a patient as set forth in claim 1,wherein stimulating the site with the electromagnetic field followingthe administration of said platelet-rich plasma includes generating inthe tissue a capacitively coupled time varying electromagnetic fieldhaving a peak amplitude of from about 0.1 to about 100 millivolts percentimeter, and having a frequency from about 1 kHz to about 200 kHz. 4.The method for promoting angiogenesis at the site on a patient as setforth in claim 1, wherein stimulating the site with an electromagneticfield following the administration of said platelet-rich plasma includesgenerating a pulsed electromagnetic field having a pulse duration fromabout 10 to 2000 microseconds.
 5. The method for promoting angiogenesisat a site on a patient as set forth in claim 1, wherein promotingangiogenesis of the site upon application of said electromagnetic fieldand said platelet-rich plasma comprises: promoting the production ofFGF-2 at the site upon the application of said electromagnetic field;and upregulating the proliferation of endothelial cells at the site uponapplication of said electromagnetic field.
 6. The method for promotingangiogenesis at a site on a patient as set forth in claim 1, furthercomprising administering stem cells to the site, said stem cellsselected from the group consisting of adipose stromal cells, stem cellsderived from bone marrow aspirate, and combinations thereof.
 7. Themethod for promoting angiogenesis at a site on a patient as set forth inclaim 6, wherein administering stem cells to the site comprises:performing lipoaspiration on the patient to obtain adipose tissue, saidadipose tissue including adipose stem cells and adipocytes;enzymatically digesting said adipose tissue; and separating said adiposestem cells from said adipocytes in said enzymatically digested adiposetissue.
 8. The method for promoting angiogenesis at a site on a patientas set forth in claim 6, wherein administering stem cells to the sitecomprises: isolating bone marrow aspirate from the patient, said bonemarrow aspirate including stem cells; centrifuging said bone marrowaspirate to concentrate said stem cells; isolating said concentratedstem cells derived from bone marrow aspirate.
 9. The method forpromoting angiogenesis at a site on a patient as set forth in claim 1,wherein administering platelet-rich plasma to the site further comprisesadministering an isolated angiogenic factor selected from the groupconsisting of angiogenin, angiopoietin-1, del-1 protein, acidicfibroblast growth factor (aFGF or FGF-1), basic fibroblast growth factor(bFGF or FGF-2), follistatin, granulocyte colony-stimulating factor(G-CSF), hepatocyte growth factor (HGF), interleukin-8 (IL-8), leptin,midkine, placental growth factor, platelet-derived endothelial growthfactor (PD-ECGF), platelet-derived growth factor (PDGF), pleiotrophin(PTN), progranulin, proliferin, transforming growth factor alpha(TGF-α), transforming growth factor beta (TGF-β), tumor necrosis factoralpha (TNF-α), vascular endothelial growth factor (VEGF), vascularpermeability factor (VPF), and combinations thereof.
 10. The method forpromoting angiogenesis at a site on a patient as set forth in claim 1,wherein the site on the patient exhibits one of the followingconditions: coronary artery disease, stroke, delayed wound healing,chronic wound, peripheral vascular disease, ischemia, chronictendonosis, wound or tissue breakdown resulting from radiationtreatment, and myocardial infarction.
 11. The method for promotingangiogenesis at a site on a patient as set forth in claim 1, wherein thesite on the patient exhibits one of the following conditions: stroke,peripheral vascular disease, ischemia, and wound or tissue breakdownresulting from radiation treatment.
 12. A method for promotingangiogenesis at the site of an ulcer on a patient comprising: obtainingblood compatible with the patient; isolating platelet-rich plasma byloading said blood into a tube having a buoy, spinning said tube so thatsaid buoy defines an interface between platelet-rich plasma andplatelet-poor plasma contained within said blood, and collecting saidplatelet-rich plasma; administering said platelet-rich plasma to thesite of the ulcer; and stimulating the site with an electromagneticfield following administration of said platelet-rich plasma to the site,thereby promoting angiogenesis at the site upon application of saidelectromagnetic field and said platelet-rich plasma to the site.
 13. Themethod for promoting angiogenesis at a site on a patient as set forth inclaim 12, wherein stimulating the site with the electromagnetic fieldfollowing the administration of said platelet-rich plasma includesgenerating in the tissue a capacitively coupled time varyingelectromagnetic field having a peak amplitude of from about 0.1 to about100 millivolts per centimeter, and having a frequency from about 1 kHzto about 200 kHz.
 14. The method for promoting angiogenesis at the siteon a patient as set forth in claim 12, wherein stimulating the site withan electromagnetic field following the administration of saidplatelet-rich plasma includes generating a pulsed electromagnetic fieldhaving a pulse duration from about 10 to 2000 microseconds.
 15. Themethod for promoting angiogenesis at a site on a patient as set forth inclaim 12, further comprising administering stem cells to the site,wherein said stem cells are selected from the group consisting ofadipose stromal cells, stem cells derived from bone marrow aspirate, andcombinations thereof.
 16. The method for promoting angiogenesis at asite on a patient as set forth in claim 15, wherein administering stemcells to the site comprises: isolating bone marrow aspirate from thepatient, said bone marrow aspirate including stem cells; centrifugingsaid bone marrow aspirate to concentrate said stem cells; isolating saidconcentrated stem cells derived from bone marrow aspirate.
 17. Themethod for promoting angiogenesis at a site on a patient as set forth inclaim 12, wherein administering platelet-rich plasma to the site furthercomprises administering an isolated angiogenic factor selected from thegroup consisting of angiogenin, angiopoietin-1, del-1 protein, acidicfibroblast growth factor (aFGF or FGF-1), basic fibroblast growth factor(bFGF or FGF-2), follistatin, granulocyte colony-stimulating factor(G-CSF), hepatocyte growth factor (HGF), interleukin-8 (IL-8), leptin,midkine, placental growth factor, platelet-derived endothelial growthfactor (PD-ECGF), platelet-derived growth factor (PDGF), pleiotrophin(PTN), progranulin, proliferin, transforming growth factor alpha(TGF-α), transforming growth factor beta (TGF-β), tumor necrosis factoralpha (TNF-α), vascular endothelial growth factor (VEGF), vascularpermeability factor (VPF), and combinations thereof.
 18. A method forpromoting angiogenesis at the site of a tissue defect in a patient,comprising: obtaining blood compatible with the patient; isolatingplatelet-rich plasma by loading said blood with a tube having a buoy,spinning said tube so that said buoy defines an interface betweenplatelet-rich plasma and platelet-poor plasma contained within saidblood, and collecting said platelet-rich plasma; administering saidplatelet-rich plasma to the site; and stimulating the site with anelectromagnetic field following administration of said platelet-richplasma to the site, thereby promoting angiogenesis at the site uponapplication of said electromagnetic field and said platelet-rich plasmato the site; wherein said tissue defect is stroke, peripheral vasculardisease, ischemia, or wound or tissue breakdown resulting from radiationtreatment.
 19. The method for promoting angiogenesis at a site on apatient as set forth in claim 18, wherein stimulating the site with theelectromagnetic field following the administration of said platelet-richplasma includes generating in the tissue a capacitively coupled timevarying electromagnetic field having a peak amplitude of from about 0.1to about 100 millivolts per centimeter, and having a frequency fromabout 1 kHz to about 200 kHz.
 20. The method for promoting angiogenesisat the site on a patient as set forth in claim 18, wherein stimulatingthe site with an electromagnetic field following the administration ofsaid platelet-rich plasma includes generating a pulsed electromagneticfield having a pulse duration from about 10 to 2000 microseconds. 21.The method for promoting angiogenesis at a site on a patient as setforth in claim 18, further comprising administering stem cells to thesite, wherein said stem cells are selected from the group consisting ofadipose stromal cells, stem cells derived from bone marrow aspirate, andcombinations thereof.
 22. The method for promoting angiogenesis at asite on a patient as set forth in claim 21, wherein administering stemcells to the site comprises: isolating bone marrow aspirate from thepatient, said bone marrow aspirate including stem cells; centrifugingsaid bone marrow aspirate to concentrate said stem cells; isolating saidconcentrated stem cells derived from bone marrow aspirate.
 23. Themethod for promoting angiogenesis at a site on a patient as set forth inclaim 18, wherein administering platelet-rich plasma to the site furthercomprises administering an isolated angiogenic factor selected from thegroup consisting of angiogenin, angiopoietin-1, del-1 protein, acidicfibroblast growth factor (aFGF or FGF-1), basic fibroblast growth factor(bFGF or FGF-2), follistatin, granulocyte colony-stimulating factor(GCSF), hepatocyte growth factor (HGF), interleukin-8 (IL-8), leptin,midkine, placental growth factor, platelet-derived endothelial growthfactor (PD-ECGF), platelet-derived growth factor (PDGF), pleiotrophin(PTN), progranulin, proliferin, transforming growth factor alpha(TGF-α), transforming growth factor beta (TGF-β), tumor necrosis factoralpha (TNF-α), vascular endothelial growth factor (VEGF), vascularpermeability factor (VPF), and combinations thereof.